Kaluza 1.2 analysis software

Peripheral blood mononuclear cells (PBMCs) were isolated using Lymphoprep (Axis Shield, Oslo, Norway) centrifugation and stored in liquid nitrogen until further use. Cells were defrozen and subsequently fixed and permeabilized using the Foxp3 / Transcription Factor Staining Buffer Set (eBioscience, San Diego, CA, USA) and stained for flow cytometric analysis with anti-CD3-PerCP (BD Biosciences, San Jose, CA, USA), anti-CD8-APC-eFluor780 (eBioscience), anti-CD127-Alexa647 (Biolegend, San Diego, CA, USA), anti-CD25-PE (eBioscience), anti-CD45RO-Alexa700 (Biolegend), anti-CD45RA-Alexa605 (BD Biosciences) and anti-FoxP3-Alexa488 (eBioscience). Cells were measured on an LSR-II flow cytometer (BD Biosciences) and the data were analyzed with Kaluza 1.2 Analysis Software (Beckman Coulter, Woerden, The Netherlands).
Absolute numbers of lymphocytes in EDTA anti-coagulated blood were determined using the BD MultiTest TruCount method with sixcolor MultiTest reagents detecting CD45, CD3, CD4, CD8, CD19, CD16 and CD56 (BD Biosciences) according to the manufacturer’s instructions. Percentages of (functional) Treg subsets were characterized as described before [7 (link),16 (link)] and CD4+ T-cell counts were used to calculate the absolute numbers of these subsets.

Apoptosis was quantified at the indicated time points after thiopurine treatment using Po-Pro staining (Cat. P3581, Molecular Probes, Eugene, OR, USA) and 7AAD staining (Cat. 559925, BD Pharmingen, San Diego, CA, USA) according to the manufacturer's instructions. Briefly, at the indicated time points, cells were harvested and washed twice with PBS, then stained in PBS with Po-Pro and 7AAD for 30 min on ice. Cells were analyzed using a Beckman Coulter Gallios flow cytometer (Beckman Coulter, Miami Lakes, FL, USA). Single positive cells for Po-Pro corresponded to the early apoptotic cells while double positive cells were considered the late apoptotic. Single positive cells for 7-AAD corresponded to the necrotic cells. The obtained data was analyzed using Kaluza 1.2 analysis software (Beckman Coulter).